Despite the small numbers of patients studied, Ga produced significant improvements in lung function in our phase 1 trial in CF adults. This improvement could be due to Ga's effects on either the bacteria or the host. This project explores the hypothesis that Ga improves lung function via anti-bacterial mechanisms. Ga has pleotrophic antimicrobial effects. As noted in the parent proposal, Ga has many iron (Fe) like features, but it cannot undergo the oxidation and reduction reactions critical for Fe's functions. Thus, Ga in enzymes renders them inactive (7, 25). In previous work (20) we showed that the opportunistic pathogen P. aeruginosa avidly takes up Ga, even when multiple Fe uptake systems are inactivated (see below). These facts suggest that Ga will have multiple antimicrobial effects, and our studies confirm this. Ga kills P. aeruginosa living in the free-living (planktonic) state, and in biofilms (20). Much lower Ga levels inhibit P. aeruginosa growth, prevent biofilm formation (20), sensitize bacteria to killing by oxidants, and disrupt bacterial Fe-starvation responses (20). Having multiple modes of action may be a hallmark of successful antibiotics. For example, aminoglycosides disrupt cell membranes and block protein synthesis (19). Quinolones and penicillins induce oxidant stress in bacteria, in addition to their conventional actions (21). Novel agents with pleotrophic effects: lessons from azythromycin. The importance of understanding Ga's in vivo mechanism(s) is exemplified by experience with another new therapy, azythromycin. Azythromycin is now used by most CF patients, and is being studied in other diseases involving infection and inflammation (15, 36). Like Ga, azythromycin has several potential therapeutic effects on the bacteria and host, including effects on macrophages, neutrophils, epithelial barrier function, anti-virulence and anti-biofilm actions, and others (15, 36). Despite widespread use, it is not known which of these effects are important in patients (15, 36). Therefore, it is unclear how azythromycin's favorable effects could be augmented, or how potential adverse effects (such as the risk of life-threatening mycobacterial infections (28)) could be limited. Understanding Ga's in vivo mechanism is important. While it will be challenging to definitively determine Ga's mechanism of action in humans, information about its in vivo activity will be valuable for several reasons. First, because Ga has potent activity in experimental infections caused by many species, understanding mechanisms of action could reveal new broad-spectrum antibacterial targets. Second, Ga's shows efficacy in biofilm infection models and in human CF, a paradigm biofilm disease (8-10, 18, 27). Understanding Ga mechanism could help identify vulnerabilities of bacteria in chronic biofilm infections. Third, understanding the in vivo mechanism of action could help us choose drugs to use in combination with Ga, as agents acting by complementary mechanisms can produce synergy.